The recent discovery of homologues of the Drosophila Toll receptor protein has elicited interest in defining the role of these proteins in innate immunity. The related Drosophila proteins Toll and 18-Wheeler are required for anti-fungal and anit-bacterial responses in the fly. The human genome encodes at least 10 distinct Toll-like receptor (TLR) protein genes although their functions in vivo are largely unknown. It has been proposed that macrophages are likely to utilize TLR proteins as part of their responses against bacterial pathogens. In mice, the mutation in a single TLR gene results in diminished responsiveness to Gram-negative bacteria both in vitro and in vivo. We have found that mice lacking functional TLR4 are highly susceptible to lethal mycobacterial infection, compared with normal mice. We recently characterized the TLR-dependent activation of macrophages by whole mycobacteria and several purified bacterial cell wall components. These studies have shown that distinct bacterial products activate cells via different TLR proteins and that different TLR agonists can elicit different patterns of cytokine production. Unlike cellular responses to Gram-negative bacteria, TLR-dependent activation of cells by Mycobacterium tuberculosis does not require CD14. Live M. tuberculosis bacilli can activate macrophages in vitro via either TLR2 or TLR4. Mycobacterial ligands responsible for cellular activation by distinct TLR-dependent pathways have been identified, but have not been biochemically characterized. The long-term objectives of this project are to characterize TLR-dependent signaling, and to define the roles that these signals play in eliciting innate immune responses in macrophages. The aims of this proposal are to (1) determine whether engagement of different TLR proteins activates both shared and distinct signal transduction pathways, (2) determine if engagement of different TLR proteins activates distinct functional response in macrophages, and (3) determine whether selective loss of TLR function results in altered innate immunity.